Field of the Invention
This invention concerns identification, isolation and partial sequencing of a cell density signaling protein produced by fibroblastic cells. In particular, the invention concerns the cell density signaling protein comprising a 14 amino acid N-terminal peptide or a fragment, variant, mutant or analog thereof, the deduced cDNA sequence from the 14 amino. acid peptide, a recombinant protein, protein and peptide-specific antibodies, and the use of the peptide and peptide-specific antibodies as therapeutic agents for regulation of cell differentiation and proliferation. The invention further concerns a method for treatment and repair of connective tissue and tendon injuries, collagen deficiency, connective tissue defects and reversal of connective tissue aging.
The level of collagen formation depends on the level of procollagen expression, a differentiated function of most fibroblastic cells in culture. The procollagen expression is under the regulation of several environmental factors, one of which is cell density.
Beginning in the early 1960""s investigators observed that an enhancement of collagen production was dependent upon an increase in cell density. However, despite its long history, little is known about the signaling mechanism that allows the cell to recognize the presence of its neighbors and translate this information into increased collagen synthesis.
Most cell types which naturally express procollagen in vivo lose this ability over time when placed in cell cultures. However, primary avian tendon (PAT) cells, when grown in a cell culture environment that is permissive for high procollagen expression, retain the potential for high levels of collagen expression, and, in this regard, demonstrate a dependency of the collagen expression on the density of the cell culture. PAT cells increase their production of procollagen in direct relation to cell density from less than 10% to about 50% of total protein synthesis, as described in Proc. Nat. Acad. Sci., 74:4453-4457 (1977).
The proliferative capacity of cells in culture is also affected by cell density. However, a definitive correlation has been difficult to obtain because cell proliferation is affected by many other cell culture parameters, only one of which is cell density. For instance, it is difficult to distinguish the effect of density per se from the possibility that cell density changes are a subset of the nutritional needs of the cell. Cell density effect may also be affected by decreased access to growth factors as cells lay down an extracellular matrix. This is supported by findings that changing the medium and even just shaking the medium above the cells is sufficient to stimulate cell division (Cell 3:207-215 (1974)).
Cell contact may also play a role in this event, as demonstrated by the fact that membrane components shed into the medium appear to inhibit cell proliferation. (Exp. Cell Research, 133:415-419 (1991))
Consequently, the relationship between cell density on the one hand and cell proliferation and the expression of differentiated function such as procollagen gene expression in the case of PAT cells, on the other hand, has been a complex problem for which it has been particularly difficult to design definitive experiments. Despite these complications, several publications describe proteins which are reported to affect density dependent cell growth. For example, a 40-45 kD density-dependent growth inhibitor, isolated from mouse 3T3 cells has been described in J. Cell. Physiol., 119:101-106 (1984); J. Cell. Physiol., 123:139-143 (1985); and J. Cell. Physiol., 130:416-419 (1987).
Cancer Res., 38:635-643 (1978), and J. Invest. Dermatol,. 87:309-312 (1986) disclose a contact inhibitory factor, isolated from hamster melanocytic cells, which restores density-dependent growth to melanoma cells. GANN Monograph on Cancer Res., 25:29-39 (1980) discloses a low molecular weight (6-8 kD) growth inhibitory factor from the cell surface of chick embryo fibroblasts.
However, despite the extensive research in the cell density and collagen expression area, the regulatory mechanism and regulatory compounds for collagen were never discovered.
In view of serious consequences of joints and tendon injuries which are connected with and are dependent on collagen formation, it would be desirable to have available means to regulate the collagen synthesis or procollagen expression and to identify their regulators.
It is, therefore, a primary objective of the current invention to isolate, identify, purify and synthesize a protein which acts as a regulator of collagen production.
All patents, patent applications and publication referred to in the specification are hereby incorporated by reference.
One aspect of the current invention is a protein produced by the fibroblastic cells acting as a cell density signal molecule able to regulate a cell differentiation and proliferation.
Another aspect of the current invention is an isolated, purified, naturally occurring or recombinantly prepared cell density signal (CDS) protein comprising a 14 amino acid peptide as its N-terminus portion, or any fragment, variant, mutant or analog of said peptide which does not change the functional characteristics of the cell density signal protein and retains the protein""s biological activity.
Still another aspect of the current invention is a peptide having an amino acid sequence Glu-Pro-Leu-Ala-Val-Val-Asp-Leu-Thr-Glu-Lys-Thr-Ile-Ser (SEQ ID NO:1) or any fragment, variant, mutant or analog thereof functioning in the same way as the whole 14 amino acid peptide wherein said peptide, fragment, variant, mutant or analog are located at N-terminus of CDS protein.
Still another aspect of the current invention is a deduced cDNA sequence encoding a peptide having an amino acid sequence Glu-Pro-Leu-Ala-Val-Val-Asp-Leu-Thr-Glu-Lys-Thr-Ile-Ser (SEQ ID NO:1) or any fragment, variant, mutant, analog thereof which expresses a peptide fragment, variant, mutant or analog functioning in the same way as the whole peptide of 14 amino acid sequence.
Still yet another aspect of the current invention is a recombinant peptide having an amino acid sequence Glu-Pro-Leu-Ala-Val-Val-Asp-Leu-Thr-Glu-Lys-Thr-Ile-Ser (SEQ ID NO:1) or any fragment, variant, mutant or analog thereof functioning in the same way as the whole 14 amino acid peptide.
Still yet another aspect of the current invention are antibodies which are specific for the cell density signal protein, 14 amino acid peptide or for any biologically active fragment, variant, mutant or analog thereof.
Another aspect of the current invention is a specific antibody recognizing a CDS protein comprising a peptide having an amino acid sequence Glu-Pro-Leu-Ala-Val-Val-Asp-Leu-Thr-Glu-Lys-Thr-Ile-Ser (SEQ ID NO:1) or a fragment, variant, mutant or analog thereof.
Still another aspect of the current invention are methods of obtaining, isolating, synthesizing and purifying cell density signal protein or 14 amino acid peptide.
Still yet another aspect of the current invention is a method for treatment of injuries and defects of connective tissue, joints and tendons.
Still another aspect of the current invention is a method for treatment of tendon and ligaments injuries and defects, said method comprising administering to a subject in need thereof a therapeutically effective amount of a protein comprising a peptide of the invention having an amino acid sequence SEQ ID NO:1, or a fragment, variant, mutant or analog thereof alone or in admixture with pharmaceutically acceptable excipients.